Discussion
The GEM surface display system has the following advantages. (1) Simple purification of a foreign protein by low-speed centrifugation is convenient and efficient. (2) Lactococcus lactis (L. lactis ) is globally recognized as a safe probiotic [20, 21]. After treatment, GEM has no safety risk as a carrier due to the absence of nucleic acids and proteins. (3) The displayed protein can effectively stimulate a stronger immune response [22]. Therefore this system has been studied and applied in many vaccine studies [15, 21, 23]. Natural PA contains three LysMs, and serine, threonine and aspartic acid are used as the interval sequences between the repeat sequences [24]. Multiple LysMs might form multivalent domains, thus increasing the anchoring activity of PA, but the anchoring activity of PA containing four LysMs was significantly reduced [13, 24]. Based on our previous research [25], we chose PA3 (containing three LysMs) as the carrier protein and an 8-amino acid sequence as the linker.
Chang [26] indicated that only the construct efficiently secreting prM/M and E antigens had the capacity to stimulate high-titered neutralizing antibodies in plasmid-vaccinated mice. Therefore, in this study, we replaced the signal peptide of ZIKV with the corresponding sequence of JEV or gp67 to realize secretory expression of target proteins. Davis [27] replaced the signal peptide of prM-E from West Nile virus (WNV) with the signal peptide of JEV, and the prediction results showed that raw cleavage site (C), signal peptide (S), and combined cleavage site (Y) scores significantly increased. Besides, the recombinant DNA vaccine could secret high levels of target proteins. Dowd [28] exchanged the signal sequence of prM-E with the analogous region of JEV to improve expression. In addition, we initially replaced the ST-TM region of ZIKV with the corresponding JEV region (data not shown). After fusion of prM-E with PA3, the target protein was hardly secreted into the supernatant and could be detected in only the sonicated supernatant. Moreover, the fusion protein with the ST-TM region could not bind to the surface of GEM (as detected by TEM, showing a smooth GEM surface, data not shown). The ST-TM region likely affected the correct expression or function of the PA3 anchor protein. Therefore, we further deleted the ST-TM region, and the fusion protein was secreted into the supernatant and successfully bound to the surface of GEM. In ZIKV DNA and virus-like particle (VLP) vaccine construction strategies, the ST-TM region is beneficial for protein expression and packaging [28-30]. However, in this study, when the target protein was co-expressed with exogenous protein PA3, deletion of the ST-TM region was beneficial for the binding of the target protein to GEM.
ZI-△-PA-GEM stimulated animals to produce specific antibodies against the ZIKV-E protein for at least 8 weeks. The PRNT50value of neutralizing antibody titer was above 1:10. There is only one serotype of ZIKV [31], and neutralizing antibody titers >10 have been found to correlate with protective efficacy [29, 32, 33]. Our results indicated that ZI-△-PA-GEM could induce potent E-specific IgG antibodies, as well as neutralizing antibody responses. In addition, the antibody subtype was Th2 biased, which might be advantageous for protection in some ways and has been reported for vaccines [25, 34-37].
As a surface molecule of B cells, CD40 is essential for the B cells isotype switching [38, 39]. The coreceptor CD19 plays an essential role in mediating the spreading of B cells and enhancing signaling through the B cell receptor (BCR) in response to membrane-bound antigen [40]. In this study, in the early stage after the first immunization, ZI-△-PA-GEM stimulated B cell activation, with an increase in CD19+ CD40+ double-positive cells. Activated B cells can produce plasma cells that generate high-affinity antibodies and memory cells, thus protecting the body against infectious diseases [41]. Moreover, ZI-△-PA-GEM recruited and/or activated DCs in secondary lymphoid organs and significantly stimulate the expression of MHC II molecules, with an increase in CD11c+ MHC II+double-positive cells. CD11c is a sign of DC maturation, and DCs are the most professional antigen-presenting cells (APCs) and the only APCs that can stimulate the initial T cell response [42]. In addition, DCs are an important factor regulating the differentiation of immature cells into Th1 or Th2 cells. MHC II molecules are important for presenting antigens on the surface of DCs [43, 44], mainly exogenous antigens, and play multiple roles in inducing protective immunity after vaccination [45]. The presentation of antigen peptides on the MHC II molecules of DCs can induce initial CD4+ T cell activation [43].
Functional regulation of Th subsets and the cytokine environment is beneficial to protective immunity [46]. ZI-△-PA-GEM induced significant cytokine secretion, including IFN-γ, IL-4, IL-6 and IL-10. Th1 cells produce important cytokines for the production of cytotoxic T cells and complement-fixing antibodies. Th2 cells produce a series of cytokines, that support antibody production [47]. The cytokines induced by vaccines are synergistic and rarely work alone, which complicates the interpretation of immune correlations [48]. The differential cytokine levels between the immunized and control groups may contribute to protection against flaviviruses [49]. Moreover, cytokines affect the differentiation of memory T cells [46]. There are two subpopulations of memory cells: effector memory T cells (migrate mainly to peripheral tissues) and TCMs (localize to the lymph nodes, blood and spleen) [46]. Here, the proportion of TCMs among splenic lymphocytes stimulated by ZI-△-PA-GEM was significantly higher, which was beneficial for rapid stimulation of the immune response during antigen stimulation.
High levels of T cell activation were also detected, especially with ZI-△-PA-GEM. Although there was no clear final conclusion concerning whether the cell-mediated immune responses against ZIKV contributed to protection, the results of a previous study support that T cells play an important role in protecting against ZIKV [3, 50]. CD8+ T cells limit the spread of viruses by recognizing and killing infected cells or secreting specific antiviral cytokines, and CD4+ T cells contribute to cytokine production and support the generation and maintenance of antibody and CD8+ T cell responses [49].
In this study, potent B cell/DC activation, cytokine responses, specific splenocytes capable of proliferation, and T cell responses, together with the production of specific antibodies, are likely to jointly contribute to protection in mice. Taken together, our data indicate that ZI-△-PA-GEM is capable of inducing humoral and cellular immune responses in mice that may protect against ZIKV infection.